Nitric oxide (.NO) is an endogenously generated, lipophilic signaling molecule that maintains vascular homeostasis via stimulation of soluble guanylate cyclase (1). In addition to mediating vascular relaxation, .NO potently modulates oxygen radical reactions, inflammatory cell function, post-translational protein modification and regulation of gene expression (2-5). There are multiple pathways whereby .NO-derived species can mediate the oxidation and nitration of biomolecules such as unsaturated fatty acids. Nitric oxide reacts at diffusion-limited rates with superoxide (O2.−, k=1.9×1010 M−1 sec−1) to yield peroxynitrite (ONOO−) and its conjugate acid, peroxynitritrous acid (ONOOH), the latter of which undergoes homolytic scission to nitrogen dioxide (.NO2) and hydroxyl radical (.OH) (2, 6). Also, biological conditions favor the reaction of ONOO− with CO2, yielding nitrosoperoxycarbonate (ONOOCO2−; k=3×104 M−1 sec−1), which rapidly yields .NO2 and carbonate (.CO3−) radicals via homolysis, or rearrangement to NO3− and CO2 (7). During inflammation, neutrophil myeloperoxidase and heme proteins such as myoglobin and cytochrome c catalyze H2O2-dependent oxidation of nitrite (NO2−) to .NO2, resulting in biomolecule oxidation and nitration that is influenced by the spatial distribution of catalytic heme proteins (8-11). Finally, even though the rate of reaction of NO with O2 is slow, (k=2×106 M−2 sec−1) the small molecular radius, uncharged nature and lipophilicity of .NO and O2 facilitate their diffusion and concentration in membranes and lipoproteins up to 20-fold (12-14). This “molecular lens” effect induced by .NO and O2 solvation in hydrophobic cell compartments accelerates the reaction of .NO with O2 to yield N2O3 and N2O4. As a result of these various reactions, a rich spectrum of primary and secondary reactions yield products capable of concerted oxidation, nitrosation and nitration of target molecules.
Multiple mechanisms can account for the nitration of fatty acids by .NO2 (15-20). During both basal cell signaling and tissue inflammatory conditions, .NO2 generated by the aforementioned reactions can react with membrane and lipoprotein lipids. Environmental sources also yield .NO2 as a product of photochemical air pollution and tobacco smoke. In both in vivo and in vitro systems, .NO2 has been shown to initiate auto-oxidation of polyunsaturated fatty acids via hydrogen abstraction from the bis-allylic carbon to form nitrous acid and a resonance-stabilized allylic radical (21). Depending on the radical environment, the lipid radical species can react with molecular oxygen to form a peroxyl radical. During inflammation or ischemia, when O2 levels are lower, lipid radicals can react to an even greater extent with .NO2 to generate multiple nitration products including singly nitrated, nitrohydroxy- and dinitro-fatty acid adducts (18, 19, 21). These products can be generated via either hydrogen abstraction or direct addition of .NO2 across the double bond. Hydrogen abstraction causes a rearrangement of the double bonds to form a conjugated diene; however, the addition of .NO2 maintains a methylene-interrupted diene configuration to yield singly nitrated polyunsaturated fatty acids (18). This arrangement is similar to nitration products generated by the nitronium ion (NO2+), which can be produced by ONOO− reaction with heme proteins or via secondary products of CO2 reaction with ONOO− (20).
Reaction of polyunsaturated fatty acids with acidified nitrite (HNO2) generates a complex mixture of products similar to those formed by direct reaction with .NO2, including the formation of singly nitrated products that maintain the bis-allylic bond arrangement (18, 19). The acidification of NO2− creates a labile species, HNO2, which is in equilibrium with secondary products, including N2O3, .NO and .NO2, all of which can participate in nitration reactions. The relevance of this pathway as a mechanism of fatty acid nitration is exemplified by physiological and pathological conditions wherein NO2− is exposed to low pH (e.g., <pH 4.0). This may conceivably occur in the gastric compartment, following endosomal or phagolysosomal acidification or in tissues following-post ischemic reperfusion.
Nitrated linoleic acid (LNO2) displays robust cell signaling activities that (at present) are anti-inflammatory in nature (20, 22-25). Synthetic LNO2 inhibits human platelet function via cAMP-dependent mechanisms (26) and inhibits neutrophil O2.− generation, calcium influx, elastase release, CD11b expression and degranulation via non-cAMP, non-cGMP-dependent mechanisms (27). LNO2 also induces vessel relaxation in part via cGMP-dependent mechanisms (22, 28). In aggregate, these data, derived from a synthetic fatty acid adduct, infer that LNO2 species represent a novel class of lipid-derived signaling mediators. To date, a gap in the clinical detection and structural characterization of nitrated fatty acids has limited defining LNO2 derivatives as biologically-relevant lipid signaling mediators that converge .NO and oxygenated lipid signaling pathways.
Therefore, it would be advantageous to produce nitrated lipids in substantially pure form so that their cell signaling activities can be characterized and their purified derivatives can be used to treat various diseases. Described herein are nitrated lipids and methods for producing nitrated lipids in pure form. Also described herein are methods for using the nitrated lipids to treat various diseases.